An emerging approach for studying protein-protein interaction in complexes is the combination of chemical crosslinking and mass spectrometric analysis of the crosslinked peptides (crosslinks) obtained after proteolysis of the complex. This approach, however, has several challenges and limitations, including the difficulty of detecting the crosslinks, the potential interference from non-informative "crosslinked peptides" (dead end and intrapeptide crosslinks), and unambiguous identification of the crosslinks by mass spectrometry. Thus, we have synthesized a isotopically-coded ethylene glycol bis(succinimidylsuccinate) derivate (D12-EGS) which contains 12 deuterium atoms for easy detection of crosslinks, when applied in a 1:1 mixture with its H12 counterpart, and is also cleavable for releasing the crosslinked peptides allowing unambiguous identification by MS sequencing. Moreover, hydrolytic cleavage permits rapid distinguishing between different types of crosslinks. Cleavage of a dead end crosslink produces a doublet with peaks 4.03 Da apart, with the lower peak appearing at a molecular mass 162 Da lower than the mass of the H12 form of the original crosslinked peptide. Cleavage of an intrapeptide crosslink leads to a doublet 8.05 Da apart, and 62 Da lower than the molecular mass of the H12 form of the original crosslinked peptide. Cleavage of an interpeptide crosslink forms a pair of 4.03-Da doublets, with the lower-mass member of each pair each shifted up from its unmodified molecular weight by 82 Da, due to the attached portion of the crosslinker. All of this information has been incorporated into a software algorithm allowing automatic screening and detection of crosslinks and crosslink types in matrix-assisted laser desorption/ionization mass spectra. In summary, the ease of detection of these species through the use of an isotopically-coded cleavable crosslinker and our software algorithm, followed by mass spectrometric sequencing of the crosslinked peptides after cleavage, has been shown to be a powerful tool for studies of multi-component protein complexes